Staphylococcus aureus (S. aureus) is a gram positive bacteria that is a common pathogen in hospitals. Infection with S. aureus often results in severe and destructive infections of the skin and soft tissues. Uncontrolled infection, especially in vulnerable patients, may spread to many tissues and organs, notably including joints, heart valves, and the lungs, often leading to sepsis and death. A significant proportion of these infections are the result of methicillin-resistant S. aureus (MRSA), and many strains are in fact resistant to multiple antibiotics. Contagious and antibiotic-resistant S. aureus strains are termed Methicillin-resistant Staphylococcus aureus (MRSA). One such strain, community-associated MRSA (CA-MRSA), has emerged as a notoriously virulent and lethal cause of pneumonia even in the young and healthy. In general, there has been a dramatic increase in the proportion of cases attributable to MRSA which is now the cause of 50% of all S. aureus infections in some intensive care units. By some estimates, 20-40% of all hospital-acquired pneumonia in the US, including ventilator-associated pneumonia (VAP), is due to MRSA, and VAP due to MRSA is associated with a poorer outcome compared to VAP with other pathogens.
In primary bacterial pneumonia, including S. aureus pneumonia, the central therapeutic focus is on killing or otherwise controlling the offending pathogen. Such interventions may take days or even weeks to be fully effective, and consequently are not optimal for rapidly countering the effects of potent and destructive virulence factors that frequently mediate pathogen-associated lung and soft tissue injury. Broad, non-specific interventions targeting the patient immune response, such as use of corticosteroids [Meijvis et al., J. Internal Med., 272: 25-35 (2012)], have been used in various pulmonary infection scenarios including pneumonia, and new approaches are being explored [Raghavendran et al., Curr. Med. Chem., 15: 1911-1924 (2008) and Darwish and Liles, Virulence, 4: 572-582 (2013)], but no general strategy for countering pathogen-associated injury has emerged.
S. aureus produces dozens of molecules known or thought to contribute to virulence. These include surface-expressed determinants such as Protein A, IsdB, clumping factor A (ClfA) and capsular polysacharrides, secreted exotoxins (including alpha, beta and gamma hemolysins), the phenol soluble modulins (PSMs), as well as other virulence factors [Otto, Annual Review of Microbiology, 64: 143-162 (2010); Wang et al., Nat. Med., 13: 1510-1514 (2007); Daum and Spellberg, Clin. Infect. Dis., 54: 560-567 (2012)]. Yet, despite the presence of so many well-characterized virulence factors, and efforts to evaluate many of these as vaccine targets [Gong et al., Clinical and Vaccine Immunology, 17:1746-1752 (2010); Arrecubieta et al., The Journal of Infectious Diseases, 198: 571-575 (2008); Anderson et al., The Journal of Infectious Diseases, 205: 1688-1696 (2012); Brady et al., Infection and Immunity, 79: 1797-1803 (2011); Joshi et al., Human Vaccines & Immunotherapeutics, 8: 336-346 (2012); Gening et al., Infection and Immunity, 78: 764-772 (2010); Cook et al., Human Vaccines, 5: 254-263 (2009)], development of an effective S. aureus vaccine has been elusive.
Alpha hemolysin or alpha toxin (Hla or AT; UniProtKB/Swiss-Prot P09616) is a protein ubiquitously secreted by most strains of S. aureus that is a critical virulence factor in S. aureus infection, host interaction, and pathology [DeLeo and Otto, J. Exp. Med., 205: 271-274 (2008); Tkaczyk et al., PLoS One, 8: e75103 (2013); Berube and Wardenburg, Toxins, 5: 1140-1166 (2013); Cho et al., J. Clin. Invest. 120: 1762-1773 (2010)] (16-19). Wild-type, methicillin-sensitive S. aureus (MSSA) and MRSA strains associated with human disease are highly virulent in mouse pneumonia, sepsis and dermonecrosis models, yet the respective hla deletion mutants are almost completely devoid of pathogenicity [Kennedy et al., J. Infect. Dis., 202: 1050-1058 (2010); Bubeck et al., Infect. Immun., 75: 1040-1044 (2007)]. Importantly, the high levels of virulence associated with CA-MRSA strains, in particular pulse field gel electrophoresis type (pulsotype) USA300, which is responsible for the preponderance of skin and soft tissue infections in otherwise healthy individuals in the community, appear to be related, in part, to the high levels of AT produced by these strains [Otto, Annual Review of Microbiology, 64: 143-162 (2010); Li et al., J. Infect. Dis., 202: 1866-1876 (2010); Li et al., Proc. Natl. Acad. Sci. USA, 106: 5883-5888 (2009)].
AT is a 293 residue protein that binds ADAM10 at the cell surface and then self-associates to form a heptameric structure that creates a pore in eukaryotic membranes [Bhakdi and Tranum-Jensen, Microbiol. Rev., 55: 733-751 (1991); Wilke and Bubeck Wardenburg, Proc. Natl. Acad. Sci. USA, 107: 13473-13478 (2010)]. The heptamer pore channel is a 14-stranded beta barrel formed by contributions of a beta-hairpin loop from each of the monomeric alpha-toxin molecules [Song et al., Science, 274: 1859-1866 (1996)]. AT is a prototypical example of a multimeric pore-forming toxin.
Studies in the early 1990s first demonstrated that passive immunization with rabbit antibody elicited to H35L, a non-toxic mutant of AT [Menzies and Kernodle, Infect. Immun., 62: 1843-1847 (1994)], is capable of mediating protection of mice from lethal S. aureus challenges in a model of lethal sepsis, and more recently, active immunization of mice with H35L has been shown to confer significant protection in the mouse models of pneumonia, sepsis and dermonecrosis [Kennedy, supra; Brady et al., PLoS One, 8: e63040 (2013); Prabhakara et al., Infect. Immun., 81: 1306-1315 (2013); Menzies and Kernodle, Infect. Immun., 64: 1839-1841 (1996); Rauch et al., Infect. Immun., 80: 3721-3732 (2012); Bubeck Wardenburg and Schneewind, J. Exp. Med., 205: 287-294 (2008)]. Passive immunization with anti-AT antibodies in a mouse model was also studied in Tkaczyk et al., Clin. Vaccine Immunol., 19(3): 377-385 (2012). An immunogen comprised of the n-terminal 62 amino acids from AT with a C-terminal His-tag was capable of eliciting neutralizing Ab and protecting mice from S. aureus challenges in pneumonia and bacteremia models [Adhikari et al., PLoS One, 7: e38567 (2012)]. U.S. Patent Publication No. 2008/0131457 filed Feb. 27, 2007 also relates to vaccines comprising an S. aureus AT antigen. Studies in other model systems where pathology is mediated by toxin elaboration, have demonstrated that the preponderance of antibody elicited through immunization with the full length toxins are mostly non-neutralizing and nonfunctional [Abboud and Casadevall, Clin. Vaccine Immunol., 15: 1115-1123 (2008); Brossier et al., Infect. Immun., 72: 6313-6317 (2004); Little et al., Infect. Immun., 56: 1807-1813 (1988); Little et al., Microbiology, 142: 707-715 (1996); Reason et al., Infect. Immun., 77: 2030-2035 (2009)].
Though immunization of mice and rabbits with H35L, or other full length forms of AT, reproducibly elicit neutralizing Ab, few neutralizing epitopes in AT have been described to date. Monoclonal antibodies that neutralize AT have been reported to bind conformational epitopes in the cap region of AT in one case, and to bind sequences yet to be fully elucidated in a second case. See, respectively, Foletti et al., J. Mol. Biol., 425: 1641-1654 (2013) and Tkaczyk (2012), supra. In a third example, a monoclonal antibody elicited to H35L protected mice in a pneumonia model, and bound a glutathione S-transferase fusion protein comprised of amino acids 1-50 from AT. The neutralizing epitope bound by the mAb was inferred to be conformational in nature, since it was incapable of binding the denatured 1-50 on western blot, and could not be defined by a panel of overlapping 15-mer peptides spanning the region [Ragle and Bubeck Wardenburg, Infect. Immun., 77: 2712-2718 (2009)].
A need in the art therefore remains for products and methods for preventing and treating S. aureus infections, especially antibiotic-resistant infections.